Fruit and vegetable cell walls are largely polysaccharide, the major components being pectin, cellulose and xylogucan (Selvendran & Robertson, IFR Report 1989). Numerous cell wall models have been proposed which attempt to incorporate the essential properties of strength and flexibility (e.g. Albersheim, 1975 Sci. Am. 232, 81-95; Albersheim, 1976 "The primary cell wall", Plant Biochemistry, 3rd Edition, Academic Press; and Hayashi, 1989, Ann. Rev. Plant Physiol. & Plant Mol. Biol. 40, 139-168).
Xyloglucans are 1,4-.beta.-glucans that are extensively substituted with .alpha.-1,6-xylosyl side chains, some of which are 1,2 .beta.-galactosylated. They are found in large amounts in the primary cell walls of dicots but also in certain seeds, where they serve different roles.
Primary cell wall xyloglucan is tightly hydrogen bonded to cellulose microfibrils and requires concentrated alkali or strong swelling agents to release it. Xyloglucan is thought to form cross-bridges between cellulose microfibrils, the cellulose/xyloglucan network forming the major load-bearing/elastic network of the wall. DCB mutated suspension culture cells (cell walls lacking cellulose) release xyloglucan into their media, suggesting that xyloglucan is normally tightly bound to cellulose.
Hydrolysis of primary cell wall xyloglucan has been demonstrated in segments of dark grown squash hypocotyls, during IAA induced growth (Wakabayashi et al., 1991 Plant Physiol. 95. 1070-1076). Endohydrolysis of wall xyloglucan is thought to contribute to the wall loosening which accompanies cell expansion (Hayashi, cited previously). The average molecular weight of xyloglucan has also been shown to decrease during tomato fruit ripening and this may contribute to the tissue softening which accompanies the ripening process (Huber, 1983 J. Amer. Soc. Hort. Sci. 108, 405-409).
Certain seeds, e.g. nasturtium, contain up to 30% by weight of xyloglucan, stored in thickened cotyledonary cell walls, which serves as a reserve polysaccharide and is rapidly depolymerised during germination.
An endo 1,4 .beta.-D glucanase which specifically acts on xyloglucan (i.e. a xyloglucanase) has been isolated and purified to apparent homogeneity from germinating nasturtium (Tropaeolum majus L.) seeds (Edwards et al., 1986 J. Biol. Chem. 261, 9494).
The purified xyloglucanase gives a single polypeptide band on SDS polyacrylamide gel electrophoresis, (apparent molecular weight, 29-31 kDa) and isoelectric focusing (isoelectric point, 5.0). The enzyme displays an absolute specificity for xyloglucan and an endo mode of action, as determined by end product analysis following hydrolysis of tamarind seed xyloglucan (Wakabayashi et al., cited above). Although the natural substrate of the enzyme is nasturtium cotyledonary reserve xyloglucan, it has also been shown to hydrolyse primary cell wall xyloglucans in vitro (Edwards et al., cited previously). At high substrate concentrations, xyloglucan endo-transglycosylase (XET) activity has been demonstrated (Fanutti et al., 1993 The Plant Journal 3, 691-700).
The nucleotide sequence of this nasturtium xyloglucanase/XET enzyme has been determined and is disclosed in International Patent Application No. WO 93/17101 and by de Silva et al., (1993 The Plant Journal 3, 701-711).
Similar enzyme activity has been detected in other plant tissue and shown to be positively correlated with growth rate in different zones of the pea stem (Fry et al., 1992 Biochem. J. 282, 821-829). It has been proposed that XET is responsible for cutting and rejoining intermicrofibrillar xyloglucan chains and that this causes the wall-loosening required for plant cell expansion. XET activity has also been demonstrated in tomato fruit (xyloglucanase apparently activatable by xyloglucan oligosaccharides) where it is reportedly highest at the 2 days post-"breaker" stage of ripening (Machlachlan & Brady, 1992 Aust. J. Plant Physiol. 19, 137-146) and may be involved in the softening process.
This application describes the isolation from tomato plants of a nucleotide sequence which encodes an XET activity.